Synthetic hydrogels engineered to promote collecting lymphatic vessel sprouting.

The lymphatic vasculature is an essential component of the body's circulation providing a network of vessels to return fluid and proteins from the tissue space to the blood, to facilitate immune ce-ll and antigen transport to lymph nodes, and to take up dietary lipid from the intestine. The development of biomaterial-based strategies to facilitate the growth of lymphatics either for regenerative purposes or as model system to study lymphatic biology is still in its nascent stages. In particular, platforms that encourage the sprouting and formation of lymphatic networks from collecting vessels are particularly underdeveloped. Through implementation of a modular, poly(ethylene glycol) (PEG)-based hydrogel, we explored the independent contributions of matrix elasticity, degradability, and adhesive peptide presentation on sprouting of implanted segments of rat lymphatic collecting vessels. An engineered hydrogel with 680 Pa elasticity, 2.0 mM RGD adhesive peptide, and full susceptibility to protease degradability produced the highest levels of sprouting relative to other physicochemical matrix properties. This engineered hydrogel was then utilized as a scaffold to facilitate the implantation of a donor vessel that functionally grafted into the host vasculature. This hydrogel provides a promising platform for facilitating lymphangiogenesis in vivo or as a means to understand the cellular mechanisms involved in the sprout process during collecting lymphatic vessel collateralization.

Effect of Human Synovial Fluid From Osteoarthritis Patients and Healthy Individuals on Lymphatic Contractile Activity.

The lymphatic system has been proposed to play a crucial role in preventing the development and progression of osteoarthritis (OA). As OA develops and progresses, inflammatory cytokines and degradation by-products of joint tissues build up in the synovial fluid (SF) providing a feedback system to exacerbate disease. The lymphatic system plays a critical role in resolving inflammation and maintaining overall joint homeostasis; however, there is some evidence that the lymphatics can become dysfunctional during OA. We hypothesized that the functional mechanics of lymphatic vessels (LVs) draining the joint could be directly compromised due to factors within SF derived from osteoarthritis patients (OASF). Here, we utilized OASF and SF derived from healthy (non-OA) individuals (healthy SF (HSF)) to investigate potential effects of SF entering the draining lymph on migration of lymphatic endothelial cells (LECs) in vitro, and lymphatic contractile activity of rat femoral LVs (RFLVs) ex vivo. Dilutions of both OASF and HSF containing serum resulted in a similar LEC migratory response to the physiologically endothelial basal medium-treated LECs (endothelial basal medium containing serum) in vitro. Ex vivo, OASF and HSF treatments were administered within the lumen of isolated LVs under controlled pressures. OASF treatment transiently enhanced the RFLVs tonic contractions while phasic contractions were significantly reduced after 1 h of treatment and complete ceased after overnight treatment. HSF treatment on the other hand displayed a gradual decrease in lymphatic contractile activity (both tonic and phasic contractions). The observed variations after SF treatments suggest that the pump function of lymphatic vessel draining the joint could be directly compromised in OA and thus might present a new therapeutic target.

Multichromatic near-infrared imaging to assess interstitial lymphatic and venous uptake in vivo.

SIGNIFICANCE: Changes in interstitial fluid clearance are implicated in many diseases. Using near-infrared (NIR) imaging with properly sized tracers could enhance our understanding of how venous and lymphatic drainage are involved in disease progression or enhance drug delivery strategies. AIM: We investigated multichromatic NIR imaging with multiple tracers to assess in vivo microvascular clearance kinetics and pathways in different tissue spaces. APPROACH: We used a chemically inert IR Dye 800CW (D800) to target venous capillaries and a purified conjugate of IR dye 680RD with 40 kDa PEG (P40D680) to target lymphatic capillaries in vivo. Optical imaging settings were validated and tuned in vitro using tissue phantoms. We investigated multichromatic NIR imaging's utility in two in vivo tissue beds: the mouse tail and rat knee joint. We then tested the ability of the approach to detect interstitial fluid perturbations due to exercise. RESULTS: In an in vitro simulated tissue environment, free dye and PEG mixture allowed for simultaneous detection without interference. In the mouse tail, co-injected NIR tracers cleared from the interstitial space via distinct routes, suggestive of lymphatic and venous uptake mechanisms. In the rat knee, we determined that exercise after injection transiently increased lymphatic drainage as measured by lower normalized intensity immediately after exercise, whereas exercise pre-injection exhibited a transient delay in clearance from the joint. CONCLUSIONS: NIR imaging enables simultaneous imaging of lymphatic and venous-mediated fluid clearance with great sensitivity and can be used to measure temporal changes in clearance rates and pathways.

Lymphatic injury alters the contractility and mechanosensitivity of collecting lymphatics to intermittent pneumatic compression.

KEY POINTS: We present the first in vivo evidence that lymphatic contraction can entrain with an external oscillatory mechanical stimulus. Lymphatic injury can alter collecting lymphatic contractility, but not much is known about how its mechanosensitivity to external pressure is affected, which is crucial given the current pressure application methods for treating lymphoedema. We show that oscillatory pressure waves (OPW), akin to intermittent pneumatic compression (IPC) therapy, optimally entrain lymphatic contractility and modulate function depending on the frequency and propagation speed of the OPW. We show that the OPW-induced entrainment and contractile function in the intact collecting lymphatics are enhanced 28 days after a contralateral lymphatic ligation surgery. The results show that IPC efficacy can be improved through proper selection of OPW parameters, and that collecting lymphatics adapt their function and mechanosensitivity after a contralateral injury, switching their behaviour to a pump-like configuration that may be more suited to the altered microenvironment. ABSTRACT: Intermittent pneumatic compression (IPC) is commonly used to control the swelling due to lymphoedema, possibly modulating the collecting lymphatic function. Lymphoedema causes lymphatic contractile dysfunction, but the consequent alterations in the mechanosensitivity of lymphatics to IPC is not known. In the present work, the spatiotemporally varying oscillatory pressure waves (OPW) generated during IPC were simulated to study the modulation of lymphatic function by OPW under physiological and pathological conditions. OPW with three temporal frequencies and three propagation speeds were applied to rat tail collecting lymphatics. The entrainment of the lymphatics to OPW was significantly higher at a frequency of 0.05 Hz compared with 0.1 Hz and 0.2 Hz (P = 0.0054 and P = 0.014, respectively), but did not depend on the OPW propagation speed. Lymphatic function was significantly higher at a frequency of 0.05 Hz and propagation speed of 2.55 mm/s (P = 0.015). Exogenous nitric oxide was not found to alter OPW-induced entrainment. A contralateral lymphatic ligation surgery was performed to simulate partial lymphatic injury in rat tails. The intact vessels showed a significant increase in entrainment to OPW, 28 days after ligation (compared with sham) (P = 0.016), with a similar increase in lymphatic transport function (P = 0.0029). The results suggest an enhanced mechanosensitivity of the lymphatics, along with a transition to a pump-like behaviour, in response to a lymphatic injury. These results enhance our fundamental understanding of how lymphatic mechanosensitivity assists the coordination of lymphatic contractility and how this might be leveraged in IPC therapy.

Lymphatic remodelling in response to lymphatic injury in the hind limbs of sheep.

Contractile activity in the lymphatic vasculature is essential for maintaining fluid balance within organs and tissues. However, the mechanisms by which collecting lymphatics adapt to changes in fluid load and how these adaptations influence lymphatic contractile activity are unknown. Here we report a model of lymphatic injury based on the ligation of one of two parallel lymphatic vessels in the hind limb of sheep and the evaluation of structural and functional changes in the intact, remodelling lymphatic vessel over a 42-day period. We show that the remodelled lymphatic vessel displayed increasing intrinsic contractile frequency, force generation and vessel compliance, as well as decreasing flow-mediated contractile inhibition via the enzyme endothelial nitric oxide synthase. A computational model of a chain of lymphatic contractile segments incorporating these adaptations predicted increases in the flow-generation capacity of the remodelled vessel at the expense of normal mitochondrial function and elevated oxidative stress within the lymphatic muscle. Our findings may inform interventions for mitigating lymphatic muscle fatigue in patients with dysfunctional lymphatics.

A novel mouse tail lymphedema model for observing lymphatic pump failure during lymphedema development.

It has been suggested that many forms of secondary lymphedema in humans are driven by a progressive loss of lymphatic pump function after an initial risk-inducing event. However, the link between pump failure and disease progression has remained elusive due to experimental challenges in the clinical setting and a lack of adequate animal models. Using a novel surgical model of lymphatic injury, we track the adaptation and functional decline of the lymphatic network in response to surgery. This model mimics the histological hallmarks of the typical mouse tail lymphedema model while leaving an intact collecting vessel for analysis of functional changes during disease progression. Lymphatic function in the intact collecting vessel negatively correlated with swelling, while a loss of pumping pressure generation remained even after resolution of swelling. By using this model to study the role of obesity in lymphedema development, we show that obesity exacerbates acquired lymphatic pump failure following lymphatic injury, suggesting one mechanism through which obesity may worsen lymphedema. This lymphatic injury model will allow for future studies investigating the molecular mechanisms leading to lymphedema development.

Entrainment of Lymphatic Contraction to Oscillatory Flow.

Lymphedema, a disfiguring condition characterized by an asymmetrical swelling of the limbs, is suspected to be caused by dysfunctions in the lymphatic system. A possible source of lymphatic dysfunction is the reduced mechanosensitivity of lymphangions, the spontaneously contracting units of the lymphatic system. In this study, the entrainment of lymphangions to an oscillatory wall shear stress (OWSS) is characterized in rat thoracic ducts in relation to their shear sensitivity. The critical shear stress above which the thoracic ducts show a substantial inhibition of contraction was found to be significantly negatively correlated to the diameter of the lymphangion. The entrainment of the lymphangion to an applied OWSS was found to be significantly dependent on the difference between the applied frequency and the intrinsic frequency of contraction of the lymphangion. The strength of the entrainment was also positively correlated to the applied shear stress when the applied shear was less than the critical shear stress of the vessel. The ejection fraction and fractional pump flow were also affected by the difference between the frequency of the applied OWSS and the vessel's intrinsic contraction frequency. The results suggest an adaptation of the lymphangion contractility to the existing oscillatory shear stress as a function of its intrinsic contractility and shear sensitivity. These adaptations might be crucial to ensure synchronized contraction of lymphangions through mechanosensitive means and might help explain the lymphatic dysfunctions that result from impaired mechanosensitivity.

Probing the effect of morphology on lymphatic valve dynamic function.

The lymphatic system is vital to the circulatory and immune systems, performing a range of important functions such as transport of interstitial fluid, fatty acid, and immune cells. Lymphatic vessels are composed of contractile walls and lymphatic valves, allowing them to pump lymph against adverse pressure gradients and to prevent backflow. Despite the importance of the lymphatic system, the contribution of mechanical and geometric changes of lymphatic valves and vessels in pathologies of lymphatic dysfunction, such as lymphedema, is not well understood. We develop a fully coupled fluid-solid, three-dimensional computational model to interrogate the various parameters thought to influence valve behavior and the consequences of these changes to overall lymphatic function. A lattice Boltzmann model is used to simulate the lymph, while a lattice spring model is used to model the mechanics of lymphatic valves. Lymphatic valve functions such as enabling lymph flow and preventing backflow under varied lymphatic valve geometries and mechanical properties are investigated to provide an understanding of the function of lymphatic vessels and valves. The simulations indicate that lymphatic valve function is optimized when valves are of low aspect ratio and bending stiffness, so long as these parameters are maintained at high enough values to allow for proper valve closing. This suggests that valve stiffening could have a profound effect on overall lymphatic pumping performance. Furthermore, dynamic valve simulations showed that this model captures the delayed response of lymphatic valves to dynamic flow conditions, which is an essential feature of valve operation. Thus, our model enhances our understanding of how lymphatic pathologies, specifically those exhibiting abnormal valve morphologies such as has been suggested to occur in cases of primary lymphedema, can lead to lymphatic dysfunctions.

The relationship between lymphangion chain length and maximum pressure generation established through in vivo imaging and computational modeling.

The intrinsic contraction of collecting lymphatic vessels serves as a pumping system to propel lymph against hydrostatic pressure gradients as it returns interstitial fluid to the venous circulation. In the present study, we proposed and validated that the maximum opposing outflow pressure along a chain of lymphangions at which flow can be achieved increases with the length of chain. Using minimally invasive near-infrared imaging to measure the effective pumping pressure at various locations in the rat tail, we demonstrated increases in pumping pressure along the length of the tail. Computational simulations based on a microstructurally motivated model of a chain of lymphangions informed from biaxial testing of isolated vessels was used to provide insights into the pumping mechanisms responsible for the pressure increases observed in vivo. These models suggest that the number of lymphangions in the chain and smooth muscle cell force generation play a significant role in determining the maximum outflow pressure, whereas the frequency of contraction has no effect. In vivo administration of nitric oxide attenuated lymphatic contraction, subsequently lowering the effective pumping pressure. Computational simulations suggest that the reduction in contractile strength of smooth muscle cells in the presence of nitric oxide can account for the reductions in outflow pressure observed along the lymphangion chain in vivo. Thus, combining modeling with multiple measurements of lymphatic pumping pressure provides a method for approximating intrinsic lymphatic muscle activity noninvasively in vivo while also providing insights into factors that determine the extent that a lymphangion chain can transport fluid against an adverse pressure gradient. NEW & NOTEWORTHY Here, we report the first minimally invasive in vivo measurements of the relationship between lymphangion chain length and lymphatic pumping pressure. We also provide the first in vivo validation of lumped parameter models of lymphangion chains previously developed through data obtained from isolated vessel testing.

Leukotriene B4 antagonism ameliorates experimental lymphedema.

Acquired lymphedema is a cancer sequela and a global health problem currently lacking pharmacologic therapy. We have previously demonstrated that ketoprofen, an anti-inflammatory agent with dual 5-lipoxygenase and cyclooxygenase inhibitory properties, effectively reverses histopathology in experimental lymphedema. We show that the therapeutic benefit of ketoprofen is specifically attributable to its inhibition of the 5-lipoxygenase metabolite leukotriene B4 (LTB4). LTB4 antagonism reversed edema, improved lymphatic function, and restored lymphatic architecture in the murine tail model of lymphedema. In vitro, LTB4 was functionally bimodal: Lower LTB4 concentrations promoted human lymphatic endothelial cell sprouting and growth, but higher concentrations inhibited lymphangiogenesis and induced apoptosis. During lymphedema progression, lymphatic fluid LTB4 concentrations rose from initial prolymphangiogenic concentrations into an antilymphangiogenic range. LTB4 biosynthesis was similarly elevated in lymphedema patients. Low concentrations of LTB4 stimulated, whereas high concentrations of LTB4 inhibited, vascular endothelial growth factor receptor 3 and Notch pathways in cultured human lymphatic endothelial cells. Lymphatic-specific Notch1-/- mice were refractory to the beneficial effects of LTB4 antagonism, suggesting that LTB4 suppression of Notch signaling is an important mechanism in disease maintenance. In summary, we found that LTB4 was harmful to lymphatic repair at the concentrations observed in established disease. Our findings suggest that LTB4 is a promising drug target for the treatment of acquired lymphedema.

Microparticle image velocimetry approach to flow measurements in isolated contracting lymphatic vessels.

We describe the development of an optical flow visualization method for resolving the flow velocity vector field in lymphatic vessels in vitro. The aim is to develop an experimental protocol for accurately estimating flow parameters, such as flow rate and shear stresses, with high spatial and temporal resolution. Previous studies in situ have relied on lymphocytes as tracers, but their low density resulted in a reduced spatial resolution whereas the assumption that the flow was fully developed in order to determine the flow parameters of interest may not be valid, especially in the vicinity of the valves, where the flow is undoubtedly more complex. To overcome these issues, we have applied the time-resolved microparticle image velocimetry (µ -PIV) technique, a well-established method that can provide increased spatial and temporal resolution that this transient flow demands. To that end, we have developed a custom light source, utilizing high-power light-emitting diodes, and associated control and image processing software. This paper reports the performance of the system and the results of a series of preliminary experiments performed on vessels isolated from rat mesenteries, demonstrating, for the first time, the successful application of the µ -PIV technique in these vessels.

Effects of dynamic shear and transmural pressure on wall shear stress sensitivity in collecting lymphatic vessels.

Given the known mechanosensitivity of the lymphatic vasculature, we sought to investigate the effects of dynamic wall shear stress (WSS) on collecting lymphatic vessels while controlling for transmural pressure. Using a previously developed ex vivo lymphatic perfusion system (ELPS) capable of independently controlling both transaxial pressure gradient and average transmural pressure on an isolated lymphatic vessel, we imposed a multitude of flow conditions on rat thoracic ducts, while controlling for transmural pressure and measuring diameter changes. By gradually increasing the imposed flow through a vessel, we determined the WSS at which the vessel first shows sign of contraction inhibition, defining this point as the shear stress sensitivity of the vessel. The shear stress threshold that triggered a contractile response was significantly greater at a transmural pressure of 5 cmH2O (0.97 dyne/cm(2)) than at 3 cmH2O (0.64 dyne/cm(2)). While contraction frequency was reduced when a steady WSS was applied, this inhibition was reversed when the applied WSS oscillated, even though the mean wall shear stresses between the conditions were not significantly different. When the applied oscillatory WSS was large enough, flow itself synchronized the lymphatic contractions to the exact frequency of the applied waveform. Both transmural pressure and the rate of change of WSS have significant impacts on the contractile response of lymphatic vessels to flow. Specifically, time-varying shear stress can alter the inhibition of phasic contraction frequency and even coordinate contractions, providing evidence that dynamic shear could play an important role in the contractile function of collecting lymphatic vessels.

Quantification of the passive and active biaxial mechanical behaviour and microstructural organization of rat thoracic ducts.

Mechanical loading conditions are likely to play a key role in passive and active (contractile) behaviour of lymphatic vessels. The development of a microstructurally motivated model of lymphatic tissue is necessary for quantification of mechanically mediated maladaptive remodelling in the lymphatic vasculature. Towards this end, we performed cylindrical biaxial testing of Sprague-Dawley rat thoracic ducts (n = 6) and constitutive modelling to characterize their mechanical behaviour. Spontaneous contraction was quantified at transmural pressures of 3, 6 and 9 cmH2O. Cyclic inflation in calcium-free saline was performed at fixed axial stretches between 1.30 and 1.60, while recording pressure, outer diameter and axial force. A microstructurally motivated four-fibre family constitutive model originally proposed by Holzapfel et al. (Holzapfel et al. 2000 J. Elast. 61, 1-48. (doi:10.1023/A:1010835316564)) was used to quantify the passive mechanical response, and the model of Rachev and Hayashi was used to quantify the active (contractile) mechanical response. The average error between data and theory was 8.9 ± 0.8% for passive data and 6.6 ± 2.6% and 6.8 ± 3.4% for the systolic and basal conditions, respectively, for active data. Multi-photon microscopy was performed to quantify vessel wall thickness (32.2 ± 1.60 µm) and elastin and collagen organization for three loading conditions. Elastin exhibited structural 'fibre families' oriented nearly circumferentially and axially. Sample-to-sample variation was observed in collagen fibre distributions, which were often non-axisymmetric, suggesting material asymmetry. In closure, this paper presents a microstructurally motivated model that accurately captures the biaxial active and passive mechanical behaviour in lymphatics and offers potential for future research to identify parameters contributing to mechanically mediated disease development.

PKC activation increases Ca²âº sensitivity of permeabilized lymphatic muscle via myosin light chain 20 phosphorylation-dependent and -independent mechanisms.

The contractile activity of muscle cells lining the walls of collecting lymphatics is responsible for generating and regulating flow within the lymphatic system. Activation of PKC signaling contributes to the regulation of smooth muscle contraction by enhancing sensitivity of the contractile apparatus to Ca(2+). It is currently unknown whether PKC signaling contributes to the regulation of lymphatic muscle contraction. We hypothesized that the activation of PKC signaling would increase the sensitivity of the lymphatic myofilament to Ca(2+). To test this hypothesis, we determined the effects of PKC activation with phorbol esters [PMA or phorbol dibutyrate (PDBu)] on the contractile behavior of α-toxin-permeabilized rat mesenteric and cervical lymphatics or the thoracic duct. The addition of PMA or PDBu induced a significant increase in the contractile force of submaximally activated α-toxin-permeabilized lymphatic muscle independent of a change in intracellular Ca(2+) concentration, and the Ca(2+)-force relationship of lymphatic muscle was significantly left shifted, indicating greater myofilament Ca(2+) sensitivity. Phorbol esters increased the maximal rate of force development, whereas the rate of relaxation was reduced. Western blot and immunohistochemistry data indicated that the initial rapid increase in tension development after stimulation by PDBu was associated with myosin light chain (MLC)20 phosphorylation; however, the later, steady-state Ca(2+) sensitization of permeabilized lymphatic muscle was not associated with increased phosphorylation of MLC20 at Ser(19), 17-kDa C-kinase-potentiated protein phosphatase-1 inhibitor at Thr(38), or caldesmon at Ser(789). Thus, these data indicate that PKC-dependent Ca(2+) sensitization of lymphatic muscle may involve MLC20 phosphorylation-dependent and -independent mechanism(s).

Impairments in the intrinsic contractility of mesenteric collecting lymphatics in a rat model of metabolic syndrome.

Numerous studies on metabolic syndrome (MetSyn), a cluster of metabolic abnormalities, have demonstrated its profound impact on cardiovascular and blood microvascular health; however, the effects of MetSyn on lymphatic function are not well understood. We hypothesized that MetSyn would modulate lymphatic muscle activity and alter muscularized lymphatic function similar to the impairment of blood vessel function associated with MetSyn, particularly given the direct proximity of the lymphatics to the chronically inflamed adipose depots. To test this hypothesis, rats were placed on a high-fructose diet (60%) for 7 wk, and their progression to MetSyn was assessed through serum insulin and triglyceride levels in addition to the expression of metabolic and inflammatory genes in the liver. Mesenteric lymphatic vessels were isolated and subjected to different transmural pressures while lymphatic pumping and contractile parameters were evaluated. Lymphatics from MetSyn rats had significant negative chronotropic effects at all pressures that effectively reduced the intrinsic flow-generating capacity of these vessels by ∼50%. Furthermore, lymphatics were remodeled to a significantly smaller diameter in the animals with MetSyn. Wire myograph experiments demonstrated that permeabilized lymphatics from the MetSyn group exhibited a significant decrease in force generation and were less sensitive to Ca(2+), although there were no significant changes in lymphatic muscle cell coverage or morphology. Thus, our data provide the first evidence that MetSyn induces a remodeling of collecting lymphatics, thereby effectively reducing their potential load capabilities and impairing the intrinsic contractility required for proper lymph flow.

Differential effects of myosin light chain kinase inhibition on contractility, force development and myosin light chain 20 phosphorylation of rat cervical and thoracic duct lymphatics.

The intrinsic contractile activity of lymphatics varies in different regions of the body. We have previously shown that cervical lymphatics possess an inherently higher frequency but lower tone at a given pressure when compared to thoracic duct lymphatics. However, the molecular mechanisms modulating the contractile characteristics of these lymphatics are not well understood. Since myosin light chain 20 (MLC(20)) phosphorylation appears to underlie the tonic component of lymphatic contraction, we hypothesized that the thoracic duct would be more sensitive to the modulation of MLC(20) phosphorylation when compared to cervical lymphatics. To test our hypothesis, the contractile activities and MLC(20) phosphorylation of thoracic duct and cervical lymphatics were determined in the absence or presence of the specific myosin light chain kinase (MLCK) inhibitor ML-7 under both isobaric and isometric conditions. Addition of ML-7 at each concentration tested led to a decrease in tone in both vessel types. While ML-7 (10(-6) m) significantly reduced the phasic contraction frequency of cervical lymphatics, it completely stopped phasic contractions of thoracic duct at that concentration. Under isometric conditions the active peak and plateau components of tension were both significantly higher in thoracic duct compared to cervical lymphatics. ML-7 (10(-5) m) significantly decreased both the active peak and plateau tensions of thoracic duct, whereas only the active peak tension of cervical lymphatics was decreased. In thoracic duct MLC(20) di-phosphorylation, but not mono-phosphorylation, was significantly decreased with increasing transmural pressure, whereas in cervical vessels only at the higher pressures tested did MLC(20) di-phosphorylation decrease. ML-7 treatment of the thoracic duct caused a significant decrease in both the mono- and di-phosphorylated forms of MLC(20). However, in cervical vessels ML-7 treatment produced an increase in the mono-phosphorylated MLC(20) form while di-phosphorylated MLC(20) was significantly decreased. These data indicate that thoracic duct has an enhanced sensitivity to MLCK inhibition when compared to cervical lymphatics and while the status of the mono- and di-phosphorylation forms of MLC(20) affects both tonic and phasic components of lymphatic contractions, the pressure-dependent changes in tonic contractions are modulated by the status of the di-phosphorylation of MLC(20) in the lymphatics.

Measuring contraction propagation and localizing pacemaker cells using high speed video microscopy.

Previous studies have shown the ability of many lymphatic vessels to contract phasically to pump lymph. Every lymphangion can act like a heart with pacemaker sites that initiate the phasic contractions. The contractile wave propagates along the vessel to synchronize the contraction. However, determining the location of the pacemaker sites within these vessels has proven to be very difficult. A high speed video microscopy system with an automated algorithm to detect pacemaker location and calculate the propagation velocity, speed, duration, and frequency of the contractions is presented in this paper. Previous methods for determining the contractile wave propagation velocity manually were time consuming and subject to errors and potential bias. The presented algorithm is semiautomated giving objective results based on predefined criteria with the option of user intervention. The system was first tested on simulation images and then on images acquired from isolated microlymphatic mesenteric vessels. We recorded contraction propagation velocities around 10 mm/s with a shortening speed of 20.4 to 27.1 µm/s on average and a contraction frequency of 7.4 to 21.6 contractions/min. The simulation results showed that the algorithm has no systematic error when compared to manual tracking. The system was used to determine the pacemaker location with a precision of 28 µm when using a frame rate of 300 frames per second.

Substance P activates both contractile and inflammatory pathways in lymphatics through the neurokinin receptors NK1R and NK3R.

OBJECTIVE: The aim of this study was to elucidate the molecular signaling mechanisms by which substance P (SP) modulates lymphatic muscle contraction and to determine whether SP stimulates both contractile as well as inflammatory pathways in the lymphatics. METHODS: A rat mesenteric lymphatic muscle cell culture model (RMLMCs) and known specific pharmacological inhibitors were utilized to delineate SP-mediated signaling pathways in lymphatics. RESULTS: We detected expression of neurokinin receptor 1 (NK1R) and neurokinin receptor 3 (NK3R) in RMLMCs. SP stimulation increased phosphorylation of myosin light chain 20 (MLC20) as well as p38 mitogen associated protein kinase (p38-MAPK) and extracellular signal regulated kinase (ERK1/2) indicating activation of both a contractile and a pro-inflammatory MAPK pathway. Pharmacological inhibition of both NK1R and NK3R significantly affected the downstream SP signaling. We further examined whether there was any crosstalk between the two pathways upon SP stimulation. Inhibition of ERK1/2 decreased levels of p-MLC20 after SP activation, in a PKC dependent manner, indicating a potential crosstalk between these two pathways. CONCLUSIONS: These data provide the first evidence that SP-mediated crosstalk between pro-inflammatory and contractile signaling mechanisms exists in the lymphatic system and may be an important bridge between lymphatic function modulation and inflammation.

Inhibition of myosin light chain phosphorylation decreases rat mesenteric lymphatic contractile activity.

Muscular lymphatics use both phasic and tonic contractions to transport lymph for conducting their vital functions. The molecular mechanisms regulating lymphatic muscle contractions are not well understood. Based on the well-established finding that the phosphorylation of myosin light chain 20 (MLC(20)) plays an essential role in blood vessel smooth muscle contraction, we investigated if phosphorylated MLC(20) (pMLC(20)) would modulate the tonic and/or phasic contractions of lymphatic muscle. The effects of ML-7, a MLC kinase inhibitor (1-10 microM), were tested on the contractile parameters of isolated and cannulated rat mesenteric lymphatics during their responses to the known modulators, pressure (1-5 cm H(2)O) and substance P (SP; 10(-7) M). Immunohistochemical and Western blot analyses of pMLC(20) were also performed on isolated lymphatics. The results showed that 1) increasing pressure decreased both the lymphatic tonic contraction strength and pMLC(20)-to-MLC(20) ratio; 2) SP increased both the tonic contraction strength and phosphorylation of MLC(20); 3) ML-7 decreased both the lymphatic tonic contraction strength and pMLC(20)-to-MLC(20) ratio; and 4) the increase in lymphatic phasic contraction frequency in response to increasing pressure was diminished by ML-7; however, the phasic contraction amplitude was not significantly altered by ML-7 either in the absence or presence of SP. These data provide the first evidence that tonic contraction strength and phasic contraction amplitude of the lymphatics can be differentially regulated, whereby the increase in MLC(20) phosphorylation produces an activation in the tonic contraction without significant changes in the phasic contraction amplitude. Thus, tonic contraction of rat mesenteric lymphatics appears to be MLC kinase dependent.